Hepatocellular carcinoma (HCC) is a world-widely known malignant tumor and found to be mainly caused by hepatitis virus infection. Accordingly, mass immunization has been implemented against hepatitis B virus (HBV), but mainly in Asia and Africa, it has been concerned that liver carcinogenesis associated with HBV will not be eradicated for many years in the future because the age of onset is 50 years or older (NPL 1). The spread of hepatitis C virus (HCV) in Japan which occurred in the 1950s and 1960s has caused rapid increase in incidence rate of HCC since the 1980s (NPL 2), and more recently, HCV infection has also spread rapidly in other countries including the United States.
As mentioned above, HCC usually develops in patients suffering from chronic hepatitis or cirrhosis associated with hepatitis virus infection. Moreover, in most of the patients, the function of liver is already decreased at the stage when HCC develops, and thus good treatment results cannot be expected unless the patients are diagnosed as cancer at an early stage. For this reason, surveillance (follow-up) of precancerous states such as chronic hepatitis and cirrhosis should be given priority, and close surveillance should be conducted particularly on patients with high risk of developing HCC to detect HCC at an early stage for surgical operations and the like, even if they are asymptomatic in clinical examination. However, close surveillance is excessively burdensome to patients having no risk of developing HCC. Accordingly, risk assessment of HCC development is essential for management of patients with chronic liver diseases such as chronic hepatitis and cirrhosis, and thus the development of its assessment has been needed.
On the other hand, alteration of DNA methylation is among the most consistent epigenetic changes observed in human multistep carcinogenesis (NPLs 3 to 4). Accumulated results of previous studies and the like suggest that alteration of DNA methylation is involved even in early and precancerous stages (NPLs 5 to 6). Also with respect to HCC development, it is found that alternation of DNA methylation, associated with splicing and/or expression abnormality of DNA methyltransferases, is already present in liver tissues with chronic hepatitis or cirrhosis, which are obtained from patients with HCC (NPLs 7 to 11).
Unlike expression of mRNA and proteins, which is susceptible to the microenvironment of cancer cells or precursor cells, alteration of DNA methylation is stably preserved in DNA double strands through covalent bonds, and is thus characterized in that even slight alteration in a precancerous state can be detected with high sensitivity. Accordingly, alteration of DNA methylation is expected to be the most suitable indicator for assessment of cancer risk (NPLs 12 to 13). In fact, the present inventors have identified 25 BAC clones, whose DNA methylation status is able to distinguish between normal liver tissues obtained from patients without HCC and noncancerous liver tissues obtained from patients with HCC in a learning cohort, by using BAC array-based methylated CpG island amplification (BAMCA, NPLs 13 and 19), which can provide an overview of the DNA methylation tendency of individual large regions in all chromosomes (NPL 18). The present inventors have proposed that the presence or absence of DNA methylation on such BAC clones is used as an indicator for assessing risk of developing HCC (PTL 1, NPLs 14 to 19).
However, with respect to the indicator, a large amount of genomic DNA is required for assessment of cancer risk because DNA inserted in the BAC clones has an average size of 170 kbp and methylated CpG sites with high diagnostic capability have not been identified. In addition, using BAMCA for such analysis requires high cost, making it difficult to apply the above indicator to practical diagnosis. Furthermore, in consideration of application to practical diagnosis, an indicator used for assessment of cancer risk may preferably have higher sensitivity and specificity (desirably, 100%) than an indicator using the BAC clones.
Thus, although there is a strong demand for a method for estimating risk of HCC with significantly high sensitivity and specificity, furthermore a method for estimating HCC risk with significantly high sensitivity and specificity while using a very small amount of genomic DNA from patients and lowering cost, such methods have not been put into practical use so far.